The invention relates generally to the field of adenoviral vectors useful in delivering genes and methods of producing same.
Adenoviruses have been described as useful viral vectors for delivery of therapeutic genes into selected host cells. Due to interest in adenoviruses as delivery vehicles, several groups have investigated the mechanisms that allow selective packaging of the adenovirus (Ad) genome into viral capsids.
Within the left end of the Ad genome, a cis-acting packaging domain has been identified [M. Grable and P. Hearing, J. Virol. 64:2047-2056 (May 1990)]. Mutants of Ad serotype 5 (Ad5) lacking this region are nonviable but can be rescued by insertion of the left-terminal 355 nt at the right end of the viral genome. The Ad5 packaging domain has been found to function in an inverted orientation, and can be moved within several hundred base pairs from its original location without a reduction in virus yield [P. Hearing et al, J. Virol., 61:2555-2558 (August 1987)].
The Ad5 packaging domain consists of at least seven elements which are functionally redundant. Four of the first five elements contain an AT-rich repeated sequence motif termed the A repeat. The fifth element does not contain any obvious primary sequence homology to the A repeat aside from the fact that it is also AT rich. With reference to the published sequences of Ad5, A repeat I is located within nt 240-248; A repeat II is located within nt 260-268; A repeat III is located within nt 302-311; A repeat IV is located within nt 313-321; A repeat V is located within nt 337-346; A repeat VI is located within nt 363 and 368 of Ad5; A repeat VII is located within nt 370-375 of Ad5 [M. Grable and P. Hearing, J. Virol, 66(2):723-731 (February 1992)]. The literature reports efforts to determine the minimum portion of the packaging domain required to package adenovirus DNA into virions. This approach is consistent with on-going efforts to maximize safety of the vectors, as well as to provide adequate space in the viral genome for heterologous gene sequences which are delivered by the adenoviral vectors.
The art continues to search for methods of optimizing production and yield of adenoviral vectors.
The present invention provides compositions and methods useful for efficiently packaging recombinant adenoviruses and producing high yields thereof. The invention involves engineering recombinant adenoviruses to contain multiple functional adenoviral packaging domains. Suitably, these vectors contain at least five of the xe2x80x9cAxe2x80x9d repeat elements of the adenoviral packaging domains, in duplicate. Most preferably, the vectors contain at least one intact adenoviral packaging domain and a second adenoviral packaging domain containing at least five xe2x80x9cAxe2x80x9d repeat elements.
Thus, in one aspect, the invention provides (a) an adenovirus 5xe2x80x2 inverted terminal repeat, (b) a first adenovirus packaging domain, (c) a second adenovirus packaging domain; (d) a selected transgene under the control of regulatory sequences directing expression of the transgene, and (3) a 3xe2x80x2 inverted terminal repeat. Suitably, the second packaging domain is located 5xe2x80x2 to the native E1 region and the selected transgene.
In another aspect, the invention provides a pharmaceutical composition comprising a recombinant adenovirus of the invention and a physiologically compatible carrier.
In still another aspect, the invention provides a method of delivering a transgene to a selected host cell by infecting said cell with a recombinant adenovirus of the invention.
In a further aspect, the invention provides a method of increasing the packaging and yield of a selected recombinant adenovirus. The method involves engineering the selected recombinant adenovirus vector to contain at least two adenoviral packaging domains.
In yet a further aspect, the invention provides a method of producing a recombinant adenovirus which lacks functional adenoviral early, intermediate and late genes. The method involves co-culturing in a host cell (a) a recombinant adenoviral plasmid comprising multiple adenoviral packaging domains and a selected transgene, said plasmid lacking functional adenoviral early, intermediate and late genes; and (b) a helper virus. The rAd plasmids and helper virus, together with the host cell, provide sufficient adenoviral gene functions to permit packaging of the recombinant adenoviral plasmid into an adenoviral capsid.
Other aspects and advantages of the invention will be readily apparent from a review of the following detailed description of the invention.